SPEReference Manual & Users Guide

Transcription

1 SPEReference Manual & Users Guide

2 Reference Manual & Users Guide 3 SPE Table of Contents Page No. 1. Objective and Purpose of This Guide 1 2. Solid Phase Extraction Overview 1 3. The Goal of Sample Preparation 1 4. SPE vs. LLE 2 5. SPE Sorbents 2 A. Sorbent Properties... 2 B. Sorbent Specificity... 2 C. Silica-based Sorbents... 2 D. Polymeric Sorbents... 3 E. Surface Area... 4 F. Pore Size... 4 G. Particle Size SPE and Liquid Chromatography 4 7. Extraction Mechanisms 4 A. Reversed Phase... 4 B. Normal Phase... 4 C. Ion Exchange... 5 D. Sample Composition Affects the Extraction Mechanism... 5 E. Retentive vs. Non-Retentive SPE Steps in SPE Protocols 6 A. Conditioning Step... 6 B. Sample Loading Step... 7 C. Wash Step... 7 D. Elution Step Method Development and Optimization 8 A. Sorbent Chemistry Sample/Matrix Effects Analyte Structure and Properties Sorbent Specificity SPE Method Development with HPLC B. Sorbent Mass C. Product Configuration and Size Syringe Barrel Columns Cartridges Well Plates Disc Membranes SPE Manifolds Batch Processing and Automation Standard SPE Protocols 15 A. Reversed Phase Extraction Protocol B. Normal Phase Extraction Protocol C. Ion Exchange Extraction Protocols Appendices 31 I. Critical Factors in SPE: Guidelines for Product Selection II. Applications for Specific Analytes III. SPE Troubleshooting Guide IV. Literature References and Suggested Reading V. Glossary Supplement SPE Sorbent Selection Tree Poster

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4 1. OBJECTIVE AND PURPOSE OF THIS GUIDE The objective of this manual is to provide analytical chemists with a practical bench-top guide to SPE. The goal is to teach the basic principles of SPE, and facilitate the process of choosing the proper sorbent, optimizing methods, and troubleshooting problems. The initial sections of this guide contain general information about SPE sorbents, extraction mechanisms, and important background information on factors that should be considered before choosing a product or developing a method. The Supplement contains the SPE Sorbent Selection Tree in the form of a poster that allows the chemist to choose the proper SPE column and sample pretreatment based on the most basic information about the target analyte(s) and the sample/matrix. The final sections of this guide include standardized, generic procedures that can be used once the sorbent has been chosen. Also included throughout this guide are practical and helpful hints for optimizing and customizing these basic protocols for each particular target analyte(s) and sample/matrix. Finally, the last section (Appendix III) contains a comprehensive SPE Troubleshooting Guide that can be used to resolve virtually any potential difficulty that may arise with any particular sample, analyte(s), column, or method. When used in conjunction with other guidebooks from Phenomenex for Liquid and Gas chromatography (see Appendix IV), this manual allows the chromatographer to develop, optimize and solve any separation problem, regardless of its complexity, and streamline the entire analytical procedure from sample preparation through the final analysis. Phenomenex offers this SPE Users Guide as a practical aide for SPE method development, optimization, and troubleshooting. For a theoretical discussion of SPE, and chromatographic theory in general, we suggest that the reader consult the literature references (1-4) in Appendix IV. of choice for discriminating chromatographers around the world. The efficacy and economy of SPE is now well documented in a staggering number of peer-reviewed journal articles and reviews. Literally, thousands of SPE applications are now available for the extraction of key analytes in the pharmaceutical, clinical/toxicological, environmental, and biomedical fields, and new methods continue to be developed on a daily basis (see Appendix II). 3. THE GOAL OF SAMPLE PREPARATION The ultimate goal of any sample preparation technique is to facilitate analysis and maximize sample throughput by concentrating the target analyte and removing impurities. SPE accomplishes these goals because it: 1. Eliminates otherwise co-eluted impurities and/or instrument-fouling particulates. 2. Concentrates analytes and improves sensitivity (and reduces LOD and LOQ). 3. Facilitates the rapid and efficient, simultaneous processing of multiple samples (either manually or via automation). 4. Enables solvent switching or buffer exchange prior to analysis. 5. Provides high extraction efficiencies, with quantitative recoveries of analytes and low levels of contaminants. 6. Provides consistent, reproducible results (provided that the extraction conditions are properly optimized and the sorbent surface chemistry is reproducible). By combining the tremendous separation power of classical liquid chromatography with a low cost, convenient, disposable cartridge, SPE effectively achieves these goals in a more direct, economical, and reproducible manner than traditional liquid: liquid extraction (LLE). Quite frankly, SPE simply works. 1 SPE 2. SOLID PHASE EXTRACTION OVERVIEW Solid Phase Extraction (SPE) is a method for rapid sample preparation in which a solid stationary phase is typically packed into a syringe barrel (Fig.1) and used to selectively extract, concentrate, and purify target analytes prior to analysis by HPLC or GC. During the last two decades, SPE has steadily gained acceptance within the analytical community and is now rapidly replacing traditional liquid:liquid extraction (LLE) as the sample preparation technique Figure 1. Typical SPE Column See glossary (pg.59) for definition

5 SPE 2 4. SPE VS. LLE Since the mid-1970 s when commercial SPE devices were first introduced, analysts have been switching from LLE to SPE for the simple reason that it provides better results with fewer complications. In fact, SPE is currently approved as an alternative to LLE for 22 of the official methods for the US EPA (United States Environmental Protection Agency). SPE provides a number of critical benefits over LLE, including the following: 1. Improved selectivity and specificity. 2. Higher recoveries. 3. Enhanced removal of interferences and particulates. 4. Greater reproducibility. 5. Reduced labor, ease of use, and the ability to rapidly process multiple samples and facilitate automation. 6. Greater flexibility in terms of solvent miscibility. 7. Elimination of emulsions. 8. Enormous decreases in solvent consumption and a concomitant reduction in hazardous waste. 9. More environmentally-friendly solvents, and reduced exposure of lab personnel to toxic and/or flammable solvents. 10. Concentration of key analytes, enhanced analytical sensitivity, reduced LOD and LOQ, improved analytical column longevity and resolution, and minimized instrument down time. 11. Ability to optimize separations using wellestablished principles of chromatographic theory and generic HPLC method optimization techniques. Clearly, SPE offers a variety of key technical, performance and economic benefits that classical LLE cannot match. SPE consistently provides cleaner extracts and recoveries that are significantly higher than those achieved with LLE, particularly with difficult-to-extract amphoteric and highly polar, water-soluble analytes. 5. SPE SORBENTS The key element to any SPE product is the sorbent. The physicochemical properties of the sorbent determine extraction efficiency and the overall quality of the separation. A. Sorbent Properties SPE sorbents are available in a wide range of surface chemistries, pore sizes (60, 120, 300 Å), particle sizes (10, 40, 100 µm), and base supports (silica, alumina, polymers). Table 1 summarizes the physiochemical properties of the most common SPE sorbents. The optimal sorbent for any given extraction problem is dependent upon the properties of the target analyte, the sample/matrix composition, and other factors that are discussed below. The SPE Sorbent Selection Tree (see Supplement) and the next several sections of this guide are designed to help facilitate the process of choosing the proper sorbent based on each of these important factors. B. Sorbent Specificity Ideally, SPE sorbents function with affinity-like specificity, retaining only the target analyte(s) without binding any contaminants. The eluant from this ideal SPE sorbent contains only the analyte(s) of interest and is free of any extraneous peaks. In most situations, however, typical SPE sorbents are not that selective. Unless the mobile phase and other extraction conditions have been properly optimized, or the analyte concentrations are fairly high, a wide range of contaminants can also be retained and affect the analysis. As a result, it is extremely important that the sorbent chemistry is chosen carefully, and that each of the extraction conditions (sample loading, wash, and elution steps) are properly optimized. In fact, sorbent specificity is so strongly dependent upon the extraction conditions that the sample/matrix loading conditions can even determine which chromatographic mechanism predominates on a given sorbent (see Sections 7-D and 8-B, below). Consequently, the extraction conditions help determine sorbent specificity just as much as the surface chemistry itself. Details on the relative specificities and properties of each of the most common sorbents used for SPE are provided in Sections 7, 9 and 10. C. Silica-based Sorbents Silica-based SPE sorbents are the most popular because they are rigid, inexpensive, easy to derivatize and manufacture reproducibly, stable, and immune to shrinking and swelling in common aqueous and organic solvents. The most popular SPE sorbents are chemicallymodified silica particles with functional groups covalently attached to the surface (Fig.2). Modification of the silica surface dramatically alters chromatographic selectivity. For example, bare silica is extremely polar, retaining analytes via normal phase and cation exchange mechanisms. By attaching a saturated hydrocarbon such as C18, the surface becomes hydrophobic (non-polar). Covalent attachment of a variety of other functional groups has been used to produce SPE sorbents with an enormous range of chromatographic selectivities. Upon completion of the initial bonding reaction, silicabased sorbents may still contain a small quantity of unreacted or free silanols (Fig.3). These exposed silanols provide polar, acidic patches on the chromatographic surface that are capable of binding amines via hydrogen bonding and cation exchange mechanisms. Since a number of important analytes are ionized See glossary (pg.59) for definition

6 under typical extraction conditions, interaction with these residual silanols may cause undesirable retention or low recoveries. In order to minimize these secondary interactions, the bonded phase is typically subjected to an endcapping reaction in which the residual silanols are methylated, typically with trimethylsilyl (TMS) groups. However, it should be noted that in SPE, free silanols can often be used to facilitate the retention of polar analytes on reversed phase sorbents like C18, C8 and Phenyl. Non-endcapped phases provide the analyst with a powerful separation tool due to the mixed polarity of the sorbent, which contains both hydrophobic alkyl chains and polar, acidic silanol moieties. Hydrocarbon groups on the analyte interact with the hydrophobic alkyl chains, and the polar or charged functional groups on the analyte interact with the silanols, enhancing retention and affecting selectivity, often for the better. Figure 2. Structures of Common Silica-based Sorbents C18 (polymeric) 3 SPE C18 (monomeric) D. Polymeric Sorbents Polymer-based SPE sorbents or resins are typically composed of highly cross-linked polystyrenedivinylbenzene (PSDVB or SDB). In contrast to bare silica, native SDB is non-polar, and capable of strong hydrophobic and p-p interactions. Like silica, the polarity of the SDB surface can be modified by the addition of various functional groups. The newer generations of SDB resins are highly cross-linked, with negligible shrinking and swelling, the particle sizes are carefully controlled, and the polymers are cleaned extensively in order to reduce endogenous extractable contaminants. Table 1 provides a list of the typical physical properties of common silica-based and polymerbased SPE sorbents. Figure 3. C18 Bonded Silica Containing Free Silanols Table 1. Physiochemical Properties of Typical SPE Sorbents Surface Particle Pore %Carbon %Nitrogen %Sulfur Area Range Size Range Size Range Retention Support Phase Surface Modification Range Range Range (m 2 /g) (microns) (Angstrom) Mechanism Silica C18 Octadecyl (polymeric) RP Silica C18 Octadecyl (monomeric) RP Silica C8 Octyl RP Silica PH Phenyl RP Silica CN Cyanobutyl RP + NP Silica NH2 Aminopropyl NP + IEX Silica SCX Phenylsulfonic Acid IEX Silica SAX Me 3 (propyl)ammonium Cl IEX Silica Silica Acidic, Neutral NP Alumina Alumina Acidic, Neutral, Basic NP Florisil Florisil None NP Polymer SDB None RP RP = Reversed Phase, NP = Normal Phase, IEX = Ion Exchange See glossary (pg.59) for definition

7 SPE 4 E. Surface Area The surface areas for typical SPE sorbents range from 250 to 600 m 2 /gram. In fact, the one gram of sorbent contained in the 6 cc SPE cartridge has about the same surface area as a basketball court. F. Pore Size The vast majority (98%) of the surface area of SPE bonded phases is contained inside the pores. Most SPE sorbents have a mean pore size of only 60 or 70 Å. This effectively restricts the access of macromolecules greater than 15 kilodaltons (kd), limiting their ability to interact with the sorbent. G. Particle Size Most SPE products are based on 50 µm irregular silicas. These large particles provide low pressure drops and rapid flow rates without clogging. The efficiency of such large particles is relatively low, only about 5,000 plates per meter, or 5% of those obtained with 5 µm HPLC packings. In fact, the typical SPE column only contains about 5 to 10 theoretical plates, which is less than one percent of that in a typical HPLC column. Despite these differences, however, SPE columns perform quite effectively, and this is due to the selectivity imparted by their surface chemistry, and as discussed in the following sections, the chromatographic operating conditions and a variety of optimization techniques that are typically employed. 6. SPE AND LIQUID CHROMATOGRAPHY As with any analytical technique, the true versatility of SPE can only be realized and achieved by first understanding, and then properly controlling the physiochemical interactions that may exist between the sorbent and the sample/matrix components. SPE relies upon the same basic chromatographic retention mechanisms and physicochemical interactions that are utilized in classical liquid chromatography and modern HPLC. The delicate interplay of these interactions between the analyte, sorbent and mobile phase is the driving force which is responsible for the separation power of all liquid chromatographic methods, including SPE. As in HPLC, the separations achieved with SPE can readily be optimized by the proper modification of the mobile phase conditions during the sample loading, wash, and elution steps, and by the judicious choice of the stationary phase. In practice, however, SPE and HPLC often differ strongly in terms of the harshness of the mobile phase conditions utilized, and consequently, the degree and the strength of the interactions between the solutes and the stationary phase. In contrast to analytical HPLC, SPE more closely resembles the digital type of chromatography which is typically employed in preparative LC, where on-off type of step gradient conditions are frequently employed to selectively elute the contaminants from the target compound(s). With SPE, the goal is typically to adjust the mobile phase conditions to the point at which the analyte is either fully retained or fully eluted in only a few column volumes. In contrast, analytical HPLC takes advantage of relatively subtle, partial interactions that allow each of the analytes to differentially migrate along the length of the column in order to enhance resolution. Despite these differences, however, the basic principles and interactions that govern both HPLC and SPE separations are, in fact, the same (see Section 7, below). Consequently, experienced chromatographers can readily utilize their understanding of chromatographic theory and HPLC optimization techniques in order to optimize SPE separations. 7. EXTRACTION MECHANISMS SPE sorbents are most commonly categorized by the nature of their primary interaction or retention mechanism with the analyte of interest. The three most common extraction mechanisms used in SPE are reversed phase, normal phase and ion exchange (Fig.4). Table 1 provides a summary of the most common SPE sorbents and the major retention mechanisms employed on each. Details are provided below and in Section 10. A. Reversed Phase Reversed phase extractions are commonly used to extract hydrophobic or even polar organic analytes from an aqueous sample/matrix. Hydrocarbon chains on both the analyte and the sorbent are attracted to one another by low energy van der Waals dispersion forces. Common reversed phase sorbents contain saturated hydrocarbon chains such as C18 and C8, or aromatic rings such as Phenyl (PH) or SDB. Because reversed phase extractions are relatively non-specific, a wide range of organic compounds is typically retained. As a result, it is important to optimize the extraction conditions, particularly the composition of the wash solvent (see Section 8-C, below). Analytes are typically eluted with organic solvents such as methanol or acetonitrile, in combination with water, acids, bases, or other solvents and organic modifiers. B. Normal Phase Normal phase retention mechanisms are commonly employed to extract polar analytes from non-polar organic solvents. The retention mechanism is based on hydrogen bonding, dipole-dipole and p-p interactions between polar analytes and polar See glossary (pg.59) for definition

8 stationary phases such as silica, alumina and Florisil. Highly specific normal phase extractions can be obtained by carefully optimizing the polarity of the conditioning solvent and the solvent(s) used to dilute and load the sample/matrix. Analytes can be eluted with the use of relatively low concentrations of polar organic solvents such as methanol or isopropanol, in combination with non-polar organic solvents. Figure 4. SPE Retention Mechanisms Reversed Phase Retention 5 SPE C. Ion Exchange Ion exchange mechanisms are used to extract charged analytes from low ionic strength aqueous or organic samples. Charged sorbents are used to retain analytes of the opposite charge. For example, positively charged analytes containing amines are retained on negatively charged cation exchangers such as sulfonic or carboxylic acids. In contrast, negatively charged analytes containing sulfonic acid or carboxylic acid groups are retained on positively charged anion exchangers containing any one of a variety of different amino groups. Ion exchange mechanisms rely on specific, high-energy coulombic interactions between the sorbent and the analyte. Only species of the proper charge are retained by the column, so most matrix contaminants are simply rinsed away to waste during the loading and the wash steps. For this reason, cation exchange SPE is commonly used for the extraction of basic compounds (drugs and other amines) from complex biological samples. Analytes are typically eluted with high ionic strength salts and buffers and/or strong acids or bases. Normal Phase Retention Anion Exchange Retention C18 D. Sample/Matrix Composition Affects the Extraction Mechanism The specificity of any sorbent towards a given analyte is highly dependent upon the composition of the sample/matrix and the soluents used for dilution. Changes in the polarity, aqueous:organic composition, ph and ionic strength during the sample loading step can actually determine the chromatographic retention mechanism and selectivity in terms of which solutes and analytes are, in fact, retained and recovered. Weak ion exchangers provide the most dramatic example of the effects of the loading (and the conditioning) solvent conditions on chromatographic specificity. In the presence of a non-polar organic solvent-rich sample/matrix, these sorbents function in the normal phase, retaining polar analytes via hydrogen bonding and dipole-dipole interactions (and adsorption chromatography). Alternatively, with an aqueous sample/matrix at neutral ph values (ph 6-8), charged analytes may be retained by an ion exchange mechanism. Finally, at ph extremes (<4 or >10) or in the presence of ion pair reagents or high salt concentrations, charged Cation Exchange Retention Carboxylic Acid (Deprotonated) as well as neutral analytes may be retained on the alkyl spacer arm via a reversed phase mechanism. Consequently, the same sorbent can be used to extract a wide range of different target analytes, and, depending upon the composition of the conditioning solvent and the sample/matrix, dramatic differences in selectivity, recovery and purity can be obtained. See glossary (pg.59) for definition

9 SPE 6 E. Retentive vs. Non-Retentive SPE The majority of SPE extractions are retentive since a sorbent retains the target analytes, while contaminants simply pass through the column to waste (Fig.5A). On the other hand, in a non-retentive extraction, the sorbent has no affinity for the analyte, but a high affinity for the sample contaminants. As a result, the analyte passes directly through the column without being retained, while the contaminants are bound (Fig.5B). Non-retentive extractions are particularly useful when the analyte is highly soluble in the sample/ matrix and/or the dilution solvent and can not be easily partitioned out of solution onto a solid or a liquid phase. They are also simpler, since there is no need for an elution step; the analyte(s) is collected during the loading and wash steps (Fig.5B). 8. STEPS IN SPE PROTOCOLS Sample preparation with SPE typically consists of 4 basic steps: conditioning, sample loading, wash, and elution (Table 2). As noted above, non-retentive extractions may only require 2 or 3 steps, since the analyte is effectively eluted during the sample loading and the wash steps. In contrast, retentive extractions typically consist of all 4 steps (Fig. 5A). In each step, the sample or mobile phases (which are either called wash or elution solvents) are passed through the sorbent and collected as desired. Because SPE takes advantages of the same physicochemical interactions utilized in classical liquid chromatography and HPLC, the recovery and purity of the target analyte(s) can be optimized by adjusting the composition of the solvents used during each of these steps (sample loading, wash and elution). In this manner, chromatographers can utilize their experience in HPLC in order to utilize the full power of SPE and optimize the extraction efficiency. A. Conditioning Step Prior to loading the sample/matrix, the SPE column is typically washed and wetted with methanol, isopropanol or another organic solvent(s) of intermediate polarity. This conditioning step removes trapped air, and solvates or activates the ligands on the chromatographic surface, enabling them to interact more effectively with the target analyte(s). The conditioning step often consists of two substeps: an initial solvation step (described above), followed directly by an equilibration step. The equilibration step typically employs a solvent with a composition that is similar to the sample/matrix in terms of the solvent ratio, ionic strength and ph. This solvent helps remove residual methanol remaining from the solvation step, and equilibrates the sorbent in a solvent that will maximize the interactions with the target analyte(s) in order to promote retention. The solvation step is typically followed directly by the equilibration solvent (and then, by the actual sample/ matrix) in order to prevent the sorbent bed from drying out or losing solvation due to evaporation. Specific details on the conditioning solvents used for each type of extraction mechanism (or sorbent) are provided in Section 10. In general, methanol has traditionally been the solvation/conditioning solvent of choice for reversed phase sorbents and ion exchangers. However, isopropanol is now recognized to be a bit more effective in this regard, owing to its slightly longer alkyl chain which interacts more strongly with the hydrocarbon chains on these sorbents. Figure 5. Schematic Illustrations of the Retentive and Non- Retentive SPE Mechanisms A) Retentive SPE B) Non-Retentive SPE See glossary (pg.59) for definition

10 Table 2. Four Basic Steps for Sample Preparation with SPE Step Conditioning Sample/Matrix Pretreatment and Loading Wash Elution Purpose To prepare the sorbent for effective interactions with the analyte(s) by solvation or activation of the ligands on the chromatographic surface, followed by equilibration in a solvent similar to the sample/matrix To adjust the sample/matrix composition (via dilution, etc.) such that the analyte(s) is quantitatively retained on the sorbent while the amount of bound impurities is minimized To remove impurities that are bound to the sorbent less strongly than the analyte(s) To selectively desorb and recover the analyte(s) by disrupting the analyte-sorbent interactions For normal phase or adsorption chromatography, the conditioning step often consists of a single equilibration step with a solvent system composed of mostly a non-polar organic solvent (such as hexane, toluene, methylene chloride or chloroform) containing a small percentage of a miscible polar organic solvent (such as an alcohol). In many cases, the initial solvation step with methanol or isopropanol is omitted, since these solvents are able to remove some of the absorbed water from the surface of the sorbent, and this can have a dramatic effect on selectivity (see Section 10-B-2). In general, solvent volumes that are between 2 to 4 times the sorbent bed volume are typically necessary to ensure proper conditioning, washing and elution. Conditioning with less than 2 to 4 bed volumes increases the risk of incomplete solvation of the bed and low or irreproducible recoveries, while more than 4 bed volumes is typically unnecessary. Conventional packed-bed SPE products typically have a bed volume of approximately 150 µl per 100 mg of sorbent. The densities and void volumes of SPE membranes and discs are much more variable, depending upon the loading density and the physical nature of the membrane support material (see Section 9-C). B. Sample Loading Step The sample loading step includes any sample/matrix pretreatment and/or dilution that may be required, as well as the actual application and introduction of the sample/matrix onto the SPE column. Sample pretreatment often consists of a dilution step in which the sample/matrix is mixed with a weak solvent which promotes analyte retention, as described below and in Section 10. The major goal of the sample loading step is to ensure that the target analyte(s) is quantitatively retained by the sorbent. As a result, it is imperative that the sample/ matrix composition is adjusted in a manner which facilitates the binding of the analyte(s). This may require the dilution of the sample in a weaker solvent; one with a higher polarity (water) for reversed phase, a non-polar solvent such as hexane for normal phase, or a low ionic strength, ph-adjusted buffer for ion exchange. For details, consult Section 10 or the SPE Sorbent Selection Tree in the Supplement. The second (often overlooked) goal of the sample loading step is to adjust the solvent conditions in order to minimize the number of impurities that are bound. This will often enhance the capacity of the sorbent for the target analyte, and improve purity. However, accomplishing this goal typically requires the loading conditions to be adjusted in the opposite direction of those required to facilitate the retention and recovery of the analyte (as described above). As a result, this method optimization tool is often omitted altogether, and is typically used only as a last resort in order to remove strongly retained or tenacious contaminants. Details on the exact conditions required for loading samples on various sorbents are given in Section 10, and on the SPE Sorbent Selection Tree in the Supplement. The degree of separation achieved during the sample loading step, and chromatographic specificity in general, are highly dependent upon the solvent composition of the sample/matrix (and the conditioning solvent). Changes in the polarity, aqueous:organic composition, ionic strength and/or ph of the sample/matrix as well as the conditioning solvent can each have a dramatic effect on analyte recovery as well as the level and the nature of the contaminants that are retained. As noted in Section 7- D, above, the loading conditions can actually determine the chromatographic retention mechanism that predominates on any given sorbent. C. Wash Step The wash step(s) is designed to elute impurities that are retained on the sorbent less strongly than the target analyte(s), and to rinse or push out residual, unretained sample/matrix components that may remain from the sample loading step. The ideal wash solvent removes all of these impurities without affecting the retention and subsequent recovery of the target analyte(s). Clearly, the wash step must contain a solvent of intermediate strength, not as weak as the loading solvent and not as strong as the elution solvent. Another restriction placed on the wash solvent is that it must be miscible with the diluted sample/matrix as well as the elution solvent; otherwise, the column must be thoroughly dried in between steps. 7 SPE See glossary (pg.59) for definition

11 SPE 8 The wash step typically receives the greatest attention during method development and optimization studies. This is probably because it often provides the most dramatic improvements in purity, and the effects of systematic changes in the wash solvent composition can readily be assessed. In situations in which the surface chemistry of the SPE and analytical HPLC columns are similar (e.g., both are C18), the HPLC mobile phase composition approximates the optimal SPE wash solvent. For reversed phased extractions, the wash solvent typically consists of an aqueous mixture of either acetonitrile or methanol with an organic composition between 5 and 50%. For normal phase extractions, the wash solvent typically contains a small amount of polar organic solvent added to the same non-polar organic solvent that was used to load the sample. For ion exchangers, the wash step typically consists of a buffer with a low to intermediate ionic strength. Optimal volumes for the wash step are typically around 1 ml for every 100 mg of sorbent, or about 7 sorbent bed volumes. This volume should be sufficient to provide the consistent and effective removal of the residual, unretained impurities and to elute some of the bound contaminants. Smaller elution volumes may yield inconsistent results, while significantly larger volumes are often a waste of solvent and indicate that the wash solvent is too weak to efficiently remove the contaminants in a timely manner. D. Elution step Once the more weakly retained contaminants have been washed from the sample, a strong solvent designed to disrupt the analyte-sorbent interactions is used to selectively desorb or elute the analyte from the sorbent. In many cases, the elution solvent must contain a mixture of several different solvents and/or chemicals (acid, base, etc.) in order to effectively break the primary interactions as well as any secondary retention mechanisms that may also be responsible for analyte binding. For example, in reversed phase, a combination of methanol, acetonitrile and a strong acid is often required in order to obtain quantitative recoveries for polar analytes. In normal phase, an acid may be added to the polar organic solvent. For ion exchangers, a polar organic solvent may be used in combination with a strong acid or base and/or a salt. In general, it is highly desirable to choose an elution solvent that is compatible with the final analytical method, or with any sample manipulation steps that may be required prior to the actual analysis (such as concentration, evaporation, and/or derivatization). For analytes that are destined to be analyzed by GC, volatile elution solvents are typically the most desirable. For HPLC analysis, the SPE elution solvent should be miscible with the initial mobile phase and typically must be diluted with a weaker solvent prior to injection. For details, see Section 10. The elution volume is typically kept to a minimum in order to improve the detection limits and analytical sensitivity. In general, at least 0.3 ml of elution solvent are typically required for every 100 mg of sorbent bed mass. In general, larger volumes (0.5 ml) are required in order to maximize recovery and reproducibility. It should also be noted that two elution steps composed of smaller elution volumes are typically more effective than one, particularly when the first aliquot is allowed to remain or soak in the sorbent bed prior to loading the second. Flow rates during the elution step should also be kept to a minimum in order to promote the elution process and enhance recovery. As noted above, a soak step may help provide the highest recoveries. The solubility rule of like dissolves like holds special significance in terms of which solvent systems can be successfully used for sample dilution and analyte elution, and which retention mechanisms and sorbents can be utilized effectively. This old adage can also be embellished upon for SPE elution solvents and sorbents, since in SPE, like elutes like, and like retains like. For example, a polar solvent will elute a polar analyte from a polar normal phase column. A hydrophobic, organic solvent will bring about the elution of a hydrophobic analyte from a hydrophobic reversed phase column. And mobile phases with a high ionic strength or charge will desorb ionic analytes from ion exchangers. Tables 4 and 5 contain detailed information on the eluotropic (eluting) strength, polarity index, miscibility, and solubility of all the common chromatography solvents. This information can be used to help pick one of the more appropriate elution solvents for normal phase or reversed phase SPE applications. Table 11 lists the relative strength of all common displacer counter ions for the elution of analytes from ion exchangers. In addition, Section 10- C provides valuable hints on the optimal elution conditions for each type of ion exchanger. Similar information can be found on the SPE Sorbent Selection Tree in the Supplement. 9. METHOD DEVELOPMENT AND OPTIMIZATION The major goal of method development is to optimize the extraction efficiency (maximize the recovery of the target analyte(s) and minimize the amount of co-eluted contaminants) under conditions that provide reproducible results in a See glossary (pg.59) for definition

12 simple, economic manner. Table 3 summarizes the 12 basic steps required to optimize any SPE method. Details are provided in the remainder of this section. Choosing the proper SPE product involves making four basic decisions, including the mode of analyte retention, the sorbent chemistry, the sorbent mass, and the physical configuration and design of the SPE product. A. Sorbent Chemistry The first step in developing an effective SPE method involves the determination of the most appropriate retention mechanism and the judicious choice of a sorbent. The most important considerations in the sorbent selection process are the physical nature of the sample/matrix, the chemical structure of the analyte, and the unique properties of each individual sorbent in terms of selectivity and specificity. 9 SPE Table 3. Systematic Steps for SPE Method Development: Critical Factors and Points to Consider 1. Classify the Analyte(s) A. Chemical class: inorganic, organic macromolecule, or organic small molecule B. Solubility/Insolubility: aqueous, polar organic, and/or non-polar organic solvents C. Polarity and hydrophobicity: polar, moderately polar, or non-polar D. Charge: neutral, ionic-neutralizable, or ionic-permanent E. Stability: reactive, acid or base labile, and/or prone to precipitation/aggregation F. Concentration: affects recovery and capacity 2. Classify the Sample/Matrix A. Solid: dissolution and pretreatment requirements, and type (powder, solid, or semi-soft) B. Gas: capture and solubilization requirements, and type (gaseous or aerosol) C. Liquid: dilution requirements, and type (aqueous, polar organic, or non-polar organic) D. Impurities: concentration, and type (particulates, salts, surfactants, fats, proteins, etc.) 3. Determine Analytical Technique A. Detection limits: peak purity, signal to noise ratio and resolution of co-extracted solutes B. SPE elution solvent: requirements or restrictions on analytical method and visa versa C. Purity requirements for SPE step: resolution and specificity of analytical method D. Analyte concentration: sample load (volume) required in SPE method 4. Determine Extraction Mechanism A. Analyte class and properties (see 1, above) B. Sample/matrix class and properties (see 2, above) C. Post-extraction sample manipulation requirements: evaporation, derivatization, etc. D. Analytical technique: GC or LC (reversed phase, normal phase, or ion exchange) 5. Choose Sorbent Chemistry A. Selectivity: purity of the target analyte(s) and overall specificity B. Recovery: retention without breakthrough and/or irreversible binding 6. Consider Sample/Matrix Volume A. Analyte concentration B. Sorbent capacity C. Elution volume D. Analytical detection limit 7. Choose Sorbent Mass A. Capacity and sample load B. Recovery C. Final elution volume and detection limits 8. Choose Product Configuration/Column Size A. Type (tube, cartridge, disc) B. Tube size (sample reservoir) C. Sample volume 9. Optimize Sample/Matrix Preparation A. Dilution (see 2, above) B. Pretreat to remove impurities (see 2D, above) C. Hydrolysis (for conjugates) 10. Optimize Wash Conditions 11. Optimize Elution 12. Analyze and Quantitate

13 SPE 10 Table 4. Solvent Miscibility and Physiochemical Properties Table 5. Solvent and Sorbent Polarity Chart

14 Table 5 contains detailed information on the relative polarity and hydrophobicity of most common SPE sorbents. This information can be used to select several likely sorbent candidates for the initial screening experiments in situations in which a reversed phase or a normal phase mechanism is appropriate. For ion exchange applications, the section of the SPE Sorbent Selection Tree on charged or ionizable organic analytes and Section 10-C provide details on the correct ion exchanger for each particular target analyte(s). For immobilized metal affinity chromatography applications, Table 4 on the SPE Sorbent Selection Tree in the Supplement provides details on the best sorbent to use for individual metal ions. Normal Phase Specificity: The relative polarity of the most common SPE sorbents is listed in Table 5. In general, CN is the least polar of all the sorbents used for normal phase chromatography. As a result, it is one of the most useful, since it typically provides good resolution and recoveries, even for extremely polar analytes, and it can also be used for reversed phase extractions. In contrast, SCX and SAX are the most polar sorbents and probably the least popular for normal phase SPE. Their extreme polarity in conjunction with their permanent charge makes recovery problematic under normal phase conditions, particularly for charged, polar analytes. Prior to the development of silica-based bonded phases, bare silica, alumina, Keiselgur and Florisil were the most popular sorbents for normal phase chromatography. Each one of these sorbents is moderately polar (Table 5). Florisil is still quite popular for the extraction of pesticides, and basic sorbents such as alumina are particularly useful for the retention of acidic compounds. In recent years, silica and NH2 have become the most commonly used normal phase sorbents. Both have moderate to low polarities and provide good resolution and recoveries. Silica is particularly useful for basic compounds which are strongly retained on acidic (acid-treated) silicas. Critical factors for normal phase SPE sorbents are their relative polarity, surface area, surface coverage, surface ph and water content (LOD or loss on drying); each of these must be fairly consistent from tube-to-tube and lot-to-lot in order to obtain reproducible results. Reversed Phase Specificity: The relative polarity of the most common reversed phase SPE sorbents is listed in Table 5. In general, SDB-based polymers are the most hydrophobic of all SPE stationary phases, followed closely by endcapped C18. Among the silica-based sorbents for reversed phase, hydrophobicity decreases in conjunction with the alkyl chain length. Differences in selectivity are typically observed on aliphatic and aromatic ligands of similar hydrophobicity. For example, aromatic solutes have a higher affinity for aromatic sorbents such as Phenyl (relative to C8), and SDB (relative to C18). Similar differences in selectivity can be observed as a result of endcapping. In most cases, non-endcapped phases have a higher affinity for more polar organic analytes/solutes. Even among the different C18 products that are commercially available for SPE, enormous differences in selectivity and hydrophobicity exist. Several examples are illustrated in the figures at the end of the SPE Troubleshooting Guide in Appendix III. Critical factors for the chromatographic performance of reversed phase SPE sorbents include surface area, carbon load, surface coverage, endcapping efficiency, LOD and pore size. Each of these properties must be fairly consistent from lot-to-lot in order to obtain reproducible results. Ion Exchange Specificity: Positively charged analytes containing amines are retained on negatively charged cation exchangers such as sulfonic acids (SCX, PRS) or carboxylic acids (WCX, CBA). In contrast, negatively charged analytes containing sulfonic acid or carboxylic acid groups are retained on positively charged anion exchangers. These sorbents include, 1 amines (WAX, NH2), 2 amines (PSA or PEI), 3 amines (DEAE), or 4 amines (SAX, Q, or Quat). Details on the relative specificity of each type of ion exchange sorbent are provided in Section 10-C. Ion exchange retention is based on specific, highenergy coulombic interactions between the sorbent and the analyte. Only species of the proper charge are retained by the column, so most matrix contaminants are simply rinsed away to waste during the loading and the wash steps. For this reason, cation exchange SPE is commonly used for the extraction of basic compounds (drugs and other amines) from complex biological samples. The target analytes are typically eluted with high ionic strength salts and buffers or with strong acids or bases. Critical factors for ion exchangers include their titration behavior and pka, total ion exchange capacity, surface chemistry, and counter ion content. 11 SPE See glossary (pg.59) for definition

15 SPE Sample/Matrix Effects As noted in Sections 7-D and 8-B, above, the solvent composition of the sample/matrix determines the extraction mechanism and is an important consideration in the sorbent selection process. As outlined in Tables 7, 8 and 9, and on the SPE Sorbent Selection Tree, each of the three major extraction mechanisms requires a specific set of solvent conditions for analyte retention (and elution). Polar, aqueous environments facilitate retention on reversed phase sorbents. Non-polar organic solvents enhance binding on normal phase sorbents. Low ionic strength, ph-adjusted, aqueous conditions promote retention on ion exchangers. The SPE Sorbent Selection Tree in the Supplement guides the user through a series of simple decisions based on the physicochemical nature of the analyte and the composition of the sample/matrix, ultimately leading to the identification of likely SPE mechanisms and sorbents. Although rather imposing at first glance, careful study reveals that it represents a simple, straightforward tool for choosing the proper SPE column. This user-friendly approach to the sorbent selection process can be applied to any sample/ matrix, including solids, liquids, or gases, and analytes with a wide range of chemical structures and properties. 2. Analyte Structure and Properties The chemical structure of the target analyte(s) is another critical factor used in the selection of an SPE sorbent. Chemical structure typically determines polarity, solubility and charge, and each of these are important factors that should be used as a guide for determining the most appropriate SPE mechanism for the separation problem at hand. Although detailed information on the solubility, polarity, and charge characteristics of any particular analyte can often be obtained from reference literature such as the Merck Index, the CRC Handbook of Chemistry, and the like (see References 5-9 in Appendix IV), it can easily be determined experimentally, or even approximated by simple examination of the chemical structure. For example, visual examination of the chemical structure of a given analyte will generally allow one to make a rough estimate of its relative hydrophobicity or polarity. This information, in turn, can be used to choose the appropriate retention mechanism and then predict retention behavior on a particular sorbent. For example, hydrophobic (hydrocarbonaceous) analytes have a high proportion of unsubstituted alkyl chains and/or aromatic rings and a low level of polar functional groups (hydroxyl, amino, carbonyl, etc.) and tend to bind strongly to reversed phase sorbents, but interact weakly with normal phase sorbents. In contrast, polar analytes have a higher proportion of polar functional groups on their hydrocarbon skeletons and a high affinity for normal phase sorbents, but are weakly retained on reversed phase columns. In a similar manner, solubility information about the target analyte(s) can also be useful in terms of determining the most appropriate extraction mechanism and sorbent, and the most appropriate loading and elution conditions. Although analyte solubility can also be determined quite easily through simple experimental means, it can also be approximated from chemical structure. In general, the standard solubility rule of Like Dissolves Like holds true. Tables 4 and 5 contain detailed information on the polarity, miscibility, solubility, and eluting strength of most common solvents for chromatography. Numerous examples of how solubility, charge, and polarity/hydrophobicity information can be used to select the appropriate retention/ extraction mechanism, sorbent, and solvent systems are provided in the SPE Sorbent Selection Tree in the Supplement. 3. Sorbent Specificity Even sorbents that rely on the same retention mechanism (reversed phase, normal phase, or ion exchange) often provide dramatically different extraction results. Subtle differences in the chemical structure and polarity amongst the sorbents within each of these individual groups can lead to major changes in selectivity, retention, and recovery for a given analyte. For example, C18 and Phenyl are both reversed phase sorbents, but C18 sorbents exhibit stronger affinities for analytes containing alkyl chains, while Phenyl sorbents interact more strongly with aromatic analytes. Different selectivities can also be expected with any two normal phase sorbents (for example, CN vs. NH2 vs. Si), and to a lesser extent, amongst cation exchangers or anion exchangers with differing functional groups. Even relatively minor differences in the chemical structure or other physicochemical properties of two otherwise similar sorbents can often translate into dramatic differences in extraction efficiency. This is certainly the case with different C18 sorbents, as illustrated in the figures in Appendix III. For this reason, it is important to screen several similar sorbents when developing a new method. In fact, early optimization work during the sorbent screening and method development stages can often lead to major long term benefits with respect to method reproducibility, recovery, and cleanliness.

16 Table 6. Typical Capacity and Elution Volumes for Common SPE Column Sizes 13 SPE 4. SPE Method Development with HPLC In most cases, effective SPE method development cannot be conducted without a quantitative analytical method that is capable of discriminating between the target analyte(s) and other endogenous contaminants in the sample/matrix. In many cases, this analytical method can provide valuable insights into the chromatographic retention behavior of the target analyte(s) and potential contaminants that can be useful when attempting to determine the initial extraction conditions. HPLC methods that are conducted on a column with the same surface chemistry as the SPE column (e.g., both are C18) typically provide valuable information about the conditions required during the extraction, and can be used to optimized the extraction method. In general, the mobile phase conditions required for isocratic elution from the HPLC column approximate the optimal wash solvent conditions for the SPE column. B. Sorbent Mass After the extraction mechanism and a specific sorbent chemistry have been identified, the next logical step is to choose the sorbent mass. The proper sorbent mass for a given extraction is one that provides sufficient capacity to retain both the analyte and any contaminants that may also be retained during the loading step. Choosing the proper sorbent mass is critical because insufficient sorbent leads to column overload and low or irreproducible recoveries, while an excess of sorbent increases solvent requirements and may also reduce recovery and purity. A good rule of thumb is that SPE cartridges retain a mass of solute (analyte plus retained contaminants) that is equivalent to 5% of the sorbent mass. Therefore, a 100 mg cartridge can retain approximately 5 mg of total solute mass (Table 6). Since the majority of SPE is performed on relativity dirty samples that contain a complex milieu of contaminants, it is common practice to err on the side of safety and choose a slightly larger sorbent mass. In situation in which the analyte is at extremely low concentrations and/or the detection signal is relatively weak, it may be advantageous to push the limits of the column capacity and load relatively large volumes of sample. For example, as much as 2 to 3 ml of serum, plasma or urine can typically be loaded on 100 mg of sorbent without adversely affecting recovery. In order to further improve detection limits, large sorbent masses (and sample volumes) should be employed in combination with the lowest possible volume of elution solvent. A practical technique for determining sorbent capacity is to conduct a single extraction using this general rule of thumb, and then determine recovery. For more detailed information on capacity and sorbent mass requirements, it is advisable to perform several breakthrough experiments in which either the sorbent mass is varied (e.g., 50, 100, 200, 500 mg), or, the sample/matrix volume is increased (e.g., 0.5, 1, 2, 4 ml). Analysis of the eluant for analyte recovery (and purity) will provide information on the optimal loading capacity and sorbent mass. See glossary (pg.59) for definition